extfieldsofstudy: Biology Medicine

text:
Marine mimivirus relatives are probably large algal viruses

Background Acanthamoeba polyphaga mimivirus is the largest known ds-DNA virus and its 1.2 Mb-genome sequence has revealed many unique features. Mimivirus occupies an independent lineage among eukaryotic viruses and its known hosts include only species from the Acanthamoeba genus. The existence of mimivirus relatives was first suggested by the analysis of the Sargasso Sea metagenomic data. Results We now further demonstrate the presence of numerous "mimivirus-like" sequences using a larger marine metagenomic data set. We also show that the DNA polymerase sequences from three algal viruses (CeV01, PpV01, PoV01) infecting different marine algal species (Chrysochromulina ericina, Phaeocystis pouchetii, Pyramimonas orientalis) are very closely related to their homolog in mimivirus. Conclusion Our results suggest that the numerous mimivirus-related sequences identified in marine environments are likely to originate from diverse large DNA viruses infecting phytoplankton. Micro-algae thus constitute a new category of potential hosts in which to look for new species of Mimiviridae.

Background
The discovery of Acanthamoeba polyphaga mimivirus was a significant breakthrough in the recent history of virology. Both mimivirus particle size (~750 nm) and its genetic repertoire (1.2 Mb-genome encoding 911 protein coding genes) are comparable to those of many parasitic cellular organisms [1,2]. This giant virus exhibits several genes for translation system components [3], and its particle contains both DNA and RNA molecules [2]. These features both quantitatively and qualitatively challenge the boundary between viruses and cells, and reignited a smol-dering debate about the origin of viruses and their role in the emergence of eukaryotes [4][5][6][7][8][9].
Mimivirus belongs to Nucleocytoplasmic large DNA viruses (NCLDVs) [10]. From its basal position in the phylogenetic trees based on conserved NCLDV core genes [1,2], the new "Mimiviridae" family was proposed for mimivirus [11]. NCLDVs now include Mimiviridae, Phycodnaviridae, Iridoviridae, Asfarviridae and Poxviridae. Mimivirus is the sole member of the Mimiviridae family. The lack of known close relatives of mimivirus makes it diffi-cult to build the evolutionary history of its surprising features. Is mimivirus one of many eccentric creatures in nature such as Rafflesia, a parasitic plant in southeastern Asia known for its gigantic flower [12]? Are the mimivirus extraordinary characteristics linked to the origin of eukaryotes [5]? Clearly, appraising the actual biological significance of this exceptional virus requires the isolation and characterization of additional members of the Mimiviridae family.
Mimivirus was initially isolated in amoebae sampled from the water of a cooling tower. Following the circumstances of its discovery, mimivirus was suspected to be a causative agent of pneumonia [13]. The presence of antibodies recognizing mimivirus in the sera of patients with community or hospital-acquired pneumonia was reported [14,15]. However, no serological evidence of mimivirus infection was found in hospitalized children in Austria [16] and mimivirus has never been isolated from an infected patient despite numerous attempts. In the laboratory, mimivirus appears to infect only species of Acanthamoeba [17]. Acanthamoeba are ubiquitous in nature and they have been isolated from diverse environments including freshwater lakes, river waters, salt water lakes, sea waters, soils and the atmosphere [18,19]. Mimivirus relatives might thus exist everywhere.
Ghedin and Claverie identified sequences similar to mimivirus genes in the environmental sequence library from the Sargasso Sea [20]. This strongly suggested the existence of mimivirus relatives in the sea. More recently, we found numerous additional "mimivirus-like" sequences in the much larger metagenomic data set generated by the Global Ocean Sampling Expedition (hereafter referred to as GOS data; [21]) (Monier et al., manuscript in preparation). However, the analysis of metagenomic data (i.e. short sequences from unknown and mixed organisms) provides no insights into the hosts susceptible to harbor the putative new species of Mimiviridae corresponding to these sequences.
While continually monitoring the new occurrences of mimivirus-like sequences in public databases, we recently noticed that the type B DNA polymerase (hereafter referred to as PolB) sequences of three lytic viruses from Norwegian coastal waters were very similar to the PolB sequence of mimivirus. The three viruses [CeV01 (Gen-Bank accession: ABU23716), PpV01 (ABU23718), PoV01 (ABU23717)] were isolated from diverse marine unicellular algae: Chrysochromulina ericina, Phaeocystis pouchetii and Pyramimonas orientalis, respectively [22,23]. C. ericina and P. pouchetii are both haptophytes but phylogenetically distant and classified in different orders, i.e. Prymnesiales and Phaeocystales. P. pouchetii forms dense and almost monospecific spring blooms while C. ericina thrive in mixed flagellate communities and at cell densities usually not attaining bloom levels [24,25]. P. orientalis is a prasinophyte belonging to the green algae. It has a worldwide distribution but the abundance is most often low with no significant contribution to the overall phytoplankton biomass [26,27]. The three algal viruses infecting these phytoplankters have all been classified as phycodnaviruses.
In this report, we first analyzed the distribution of mimivirus-like sequences found in the GOS data and mapped them on the mimivirus genome. We then performed phylogenetic analyses which indicated a very close relationship between the PolB sequences of mimivirus and the three algal viruses (CeV01, PpV01, PoV01), as well as with their homologs from the metagenomic data set.

Results
We first examined the presence of "mimivirus-like" sequences in the GOS data composed of 7.7 million sequencing reads. Based on a protocol similar to the one used by Ghedin and Claverie [20], we identified 5,293 open reading frames (ORFs; ≥ 60 aa) that are closely related to protein sequences encoded in the mimivirus genome. Of 911 mimivirus protein coding genes, 229 (25%) showed closely related sequences in the GOS data. The distribution of the number of GOS matches for each of the 229 mimivirus genes is highly variable ranging from 1 to 249 (ex. 249 hits for MIMI_R555 DNA repair protein). These 229 mimivirus genes are distributed widely along the chromosome, with an apparently higher concentration in the central part of the genome (Fig. 1). This part of the genome encodes many conserved genes including most of the NCLDV core genes [2]. Mimivirus possesses 26 NCLDV core genes (class I, II and III), of which 17 had close homologs in the GOS data (Table 1 and Additional File 1). Phylogenetic trees for the homologs of two class I core genes (L437, VV A32-type virion packaging ATPase; L206/L207, VV D5-type ATPase) confirmed the separate grouping of the mimivirus sequences with their closest homologs found in the GOS data ( Fig. 2) Among the translation related genes of mimivirus, mRNA cap binding protein gene (MIMI_L496) and translation initiation factor eEF-1 gene (MIMI_R624) had close homologs in the GOS data. Remarkably, 55 of the 229 mimivirus genes exhibiting a strong similarity in the GOS data, correspond to ORFans (i.e. ORFs lacking homologs in known species), further suggesting that their GOS homologs belong to viruses closely related to mimivirus.
We next selected fourteen mimivirus PolB-like GOS-ORF sequences that are long enough to be fully aligned with homologs from different viruses including three algal viruses, CeV01, PpV01 and PoV01. PolB sequences from CeV01 (GenBank: ABU23716), mimivirus [28] and Heter-  Fig. 3). Similar levels of bootstrap support were obtained by neighbor joining and maximum parsimony approaches (99% and 80%, respectively). Certain of the GOS-ORFs (nine GOS-ORFs) are more closely related to PolB's from CeV01 and/or PpV01 (bootstrap value = 100%), while others appear to be more closely related to PolB's from PoV01 and/or mimivirus. The percentage of identical amino acid residues between mimivirus PolB sequence and its GOS homologs in Figure  3 varies from 37% to 48%, suggesting a substantial level of genetic diversity of the mimivirus relatives in the sea. Mimivirus PolB sequence exhibits 41%, 31%, 45% identity with the PolB sequence of the three algal viruses CeV01, PpV01, and PoV01, respectively. The phylogenetic tree presented in Figure 3 supports the monophyletic grouping for iridoviruses (100%) as well as for poxviruses (75%). In contrast, the inclusion of the new mimiviruslike PolB sequences in the phylogenetic analysis apparently breaks the monophyletic grouping of viruses previously classified as member of the phycodnavirus family, robustly clustering the CeV01, PpV01, and PoV01 viruses with mimivirus. Previous studies have indicated a relatively close phylogenetic relationship [2] and a similarity in gene composition [10] between phycodnaviruses and mimivirus. Several phycodnaviruses exhibit the largest genome sizes (>300kb) after mimivirus [4]. Claverie et al. have hypothesized that Phycodnaviridae is a promising source of giant viruses [4]. In this study, we present phylogenetic evidence for a close relationship between the PolB sequences of three algal viruses (CeV01, PpV01, PoV01) and mimivirus, and for the segregation of these from homologs of other known viruses. PolB is one of the NCLDV core genes, and serves as a phylogenetic marker for the classification of large DNA viruses [33,34]. There now seems to be a continuum between the giant mimivirus and some algal viruses at least with respect to the sequence of this essential viral enzyme. The large genome sizes of CeV01, PpV01, and PoV01 might be another indication of their close evolutionary relationship with mimivirus. Phylogenetic classification of phycodnaviruses and mimiviruses (including the split of Phycodnaviridae or merging of Mimiviridae and Phycodnaviridae) may have to be revisited based on sequence information from other genetic markers such as major capsid proteins (Larsen et al. manuscript in preparation) and other NCLDV core genes.

Discussion
Our discovery of the close relationships among PolB sequences of mimivirus and the three algal viruses as well as their homologs from metagenomic data now sheds Mimivirus-like sequences in the GOS metagenomic data Figure 1 Mimivirus-like sequences in the GOS metagenomic data. The mimivirus-like sequences in the metagenomic data are likely to originate from large DNA viruses closely related to mimivirus, CeV01, PpV01 and PoV01. Probably, there is a substantial genetic variation among these putative viruses. The fact that the host algae of CeV01, PpV01 and PoV01 have worldwide distributions, suggests that these putative viruses might not be necessarily associated with marine amoebae, but rather to algal species closely related to C. ericina, P. pouchetii or P. orientalis.
Mimivirus was proposed to be a human pathogen causing pneumonia. However, the close relationship of mimivirus with viruses infecting phytoplankton does not favor this hypothesis, as eukaryotic large DNA virus groups (e.g. at the level of genus) usually correspond to a relatively narrow hosts range. Given the strong cytopathic effect of mimivirus on its amoebal host and its phylogenetic affinity with certain algal viruses, we now begin to suspect that the natural reservoir of mimivirus might be some algae. Indeed, algae are frequently found together with acanthamoeba, in anthropogenic ecosystems such as air-conditioning units.

Mimivirus Phycodnaviruses
Mimi-like metagenomic sequences the numerous "mimivirus-like" sequences detected in marine metagenomic data might originate from viruses infecting phytoplankton species related to C. ericina, P. pouchetii or P. orientalis, rather than marine amoebae. These results imply new approaches in attempting the isolation of additional, and eventually closer, relatives of mimivirus.

Methods
The scaffold sequences for the combined assembly of the GOS metagenomic data were downloaded from the CAM-ERA web site [35]. We extracted 21,406,171 ORFs (≥ aa) from the scaffolds using the EMBOSS/getorf program [36].
We defined "mimivirus-like ORFs" based on the following two-way BLASTP searches [37]. First, the amino acid sequences of the ORFs were searched against the UniProt sequence database release 11.3 (as of July 2007, [38]) using BLASTP (E-value < 0.001). This search resulted in 6,212 ORFs with its best hit to a mimivirus protein in the database. For each of the 6,212 ORFs, we extracted a segment of the mimivirus sequence that was aligned with the ORF by BLASTP. Next, this partial mimivirus sequence was searched against the UniProt database (excluding mimivirus entries in the database). If the best score obtained by this second BLASTP search is lower than the BLASTP score obtained by the first BLASTP search, we kept the ORF as "mimivirus-like". Accordingly, we obtained 5,293 mimivirus-like ORFs. The UniProt database does not contain the three entries used for the phylogenetic study (i.e. ABU23716, ABU23717, ABU23718).
Mimivirus ORFans were defined by the lack of detectable homologs in the UniProt database using BLASTP with an E-value threshold of 0.001.
Multiple sequence alignment was constructed using MUS-CLE [39]. All the gap-containing sites in the alignment were excluded in the phylogenetic analysis. We used only the polymerase domain sequences, and removed exonuclease domain sequences. The delineation of the polymerase domains were performed using the Pfam entry PF00136 [40]. Intein sequences were also removed from Mimivirus, HaV, CeV01 PolB sequences. Maximum likelihood phylogenetic analysis was performed using PhyML [41] with JTT substitution model and 100 bootstrap replicates. Neighbor joining analysis was performed using BIONJ [42]. The above methods are available from the Phylogeny.fr server [43]. Maximum parsimony analysis was performed using PHYLIP/PROTPARS [44].


s2fieldsofstudy: Biology Medicine

text:
Comparing methods of detection and quantitation of RNA editing of rat glycine receptor alpha3.

Background: Recently, it has become evident that RNA editing-related changes are important in the modulation of neuronal information processing. Alternatively edited transcripts, when meagerly present, are hard to detect. Significant functional consequences may result, however, from small differences in editing efficiency. Moreover, it is difficult to evaluate the ratio of edited transcripts. The glycine receptor alpha3 subunit (GlyR alpha3) is expressed in the spinal cord, and transcripts of GlyR alpha3 are susceptible to RNA editing. The physiological role of this editing is still unclear. To analyze changes in RNA editing in various animal models, we need reliable and practical ways to detect and quantitate GlyR alpha3 RNA editing. Results: We identified and assessed different ways of detecting edited RNA transcripts, including direct sequencing, denaturing high performance chromatography (DHPLC), allele-specific real-time PCR with TaqMan probes, and PCR with allele-specific primers. Using PCR with allele-specific primers on standard PCR products for edited and nonedited GlyR alpha3, we were able to detect as little as a 0.5% incidence of edited transcripts. We were able to detect a 5% incidence of RNA editing using direct sequencing and 2% using DHPLC. We could accurately determine the ratio of edited to non-edited RNA using DHPLC, direct sequencing, and allele-specific real-time PCR with TaqMan probes. Conclusion: We demonstrated exact and sensitive methods of detecting RNA editing. In prepared samples, we showed means of quantitating the incidence of editing of a particular site. The demonstrated methodologies should be very useful when extended to the evaluation of other types of RNA editing and single base mutations.

Introduction
RNA editing is broadly defined as any RNA processing event (excluding RNA splicing) that generates an RNA message with a nucleotide sequence that differs from corresponding genomic DNA. Recently, it has become evident that RNA editing plays an important role in the modulation of neuronal information processing, and that small differences in editing efficiency can have significant functional consequences. Mammalian RNA editing generally occurs by either cytosine-to-uracil (C-to-U) or adenosine-to-inosine (A-to-I) deamination. Good examples of C-to-U editing include conversions, mediated by a citidine deaminase in conjunction with additional cellular machinery, within apolipoprotein B RNA [1] and neurofibromin [2] . Elsewhere, involving alteration of the mRNA of glutamate and serotonin receptors, A-to-I editing of several neuronal genes has been identified. RNA editing similarly affects potassium channels. Functional consequences include effects on calcium ion permeability (GluR6; [3]), on control of G protein activation (5HT2c; [4]), and on modulation of channel inactivation (hKv1.1; [5]). A-to-I editing is caused by the ADAR (adenosine deaminases that act on RNA) family of enzymes [6]. The occurrence of inosine in poly(A)+ mRNA has been estimated at one base in 17,000 in rat brain and one in 33,000 in rat heart [7]. Very few edited transcripts have been identified [6], and it is likely that many edited transcripts await discovery. If inosine occupies a guanosine site during the translation process, its presence can alter the encoded protein [8]. Consequently, in neuroscience, it is very important to find ways of detecting these meagerly existing edited transcripts.
The glycine receptor alpha3 subunit (GlyR al-pha3) is expressed in the spinal cord where it physiologically plays a pivotal role in prostaglandin-mediated inflammatory pain transmission in the spinal cord dorsal horn. GlyR alpha3 transcripts are susceptible to RNA editing. These edited isoforms cause physiological changes. GlyR alpha3 P185L , resulting from cytidine 554 deamination, endows neurons with high glycine sensitivity and promotes sustained chloride conductance associated with tonic inhibition. [9] The physiological role of this editing is still unclear. To analyze changes in RNA editing in various animal models, we need reliable and practical ways of detecting and quantitating GlyR alpha3 RNA editing.
More easily detected than edited RNA, single nucleotide polymorphisms (SNPs) are commonly identified by various methods. Because editing creates such subtle changes in the sequence, it is as difficult to detect edited transcripts as it is to detect micro-exons. It has been possible, however, to greatly improve assay sensitivity by using PCR products as a template and sequencing with thermostable polymerases [10,11,12]. Very small amounts of mRNA can now be assayed by amplifying the template with RT-PCR, but few studies have yet presented detailed reports of the factors that contribute to the accuracy of the assay [13,14,15,16]. We tried four different methods of detecting and determining the ratio of edited-to-non-edited transcripts: DHPLC; direct sequence; and allele-specific real-time PCR with TaqMan probe as a qualitative and sensitive methods: and PCR using allele-specific primers as a sensitive method. We evaluated the four methods in detail.

Total clone making
RNA was isolated from rat spinal cord and cDNA was obtained by reverse transcription using oligo-dT. The full length cDNA encoding rat GlyR alpha3 subunit was amplified using oligonucleotides 5'-AACATGCATAATGCAACTCG-3' and 5'-CCATCCAAATGTCAATTGC-3' and subcloned into TA cloning vector pCR2.1. (Invitrogen, Tokyo, Japan). The site-directed mutation for GlyR alpha3 (P185L) was performed by QuikChange mutagenesis kit (Stratagene Tokyo, Japan). We use TA Cloning kit (Invitrogen, Tokyo, Japan) for ligation and TOPO 10 (Invitrogen, Tokyo, Japan) for transformation. After purification of plasmids, we carried out direct sequencing and confirmed that the samples contained completely full-length GlyR alpha3 wild and mutant clones.

Preparation of standard samples
We performed quantitative RT-PCR of GlyR al-pha3 edited and nonedited full length samples, according to the manufacturer's protocol, with an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA) using TaqMan probes supplied by Applied Biosystems. All reactions were run in duplicate. We performed the quantitation of both edited and nonedited samples using standard curve methods. Standard samples were prepared by controlling the concentration of these two samples at same detection cycles.

Standard PCR products
We made the standard PCR products (fragment 467 bp) for edited and nonedited GlyR alpha3 with optimase polymerase (high fidelity polymerase) using oligonucleotides F>5'AATGTTCTTTATTCAATAAGGCTGA3' and R>5'ACATAGGACACCTTTGGTAAAGAA3' which flank the editing site ( Fig. 1). A 467 bp fragment was amplified in a reaction using, as recommended by the manufacturer, 1.25 units of polymerase, 0.5 μM of each primer, and a reaction buffer with Mg 2+ along with approximately 1 ng Plasmid DNA template, 200 μM of each dNTP and distilled water to a final volume of 50 μl. Cycling conditions were 1 cycle at 95°C for 5 min followed by 25 cycles of 95°C for 45 s, 55°C for 45s, 72°C for 1.5 min, and 72°C for 7 min.  . PCR using allele-specific primers. A) ASP forward primer design. Two bases (one is a site of editing) were mismatched. B) PCR using ASPs could detect GlyR alpha3 editing incidences as low as 0.5%. While the 273 bp band for Lane 1 is clearly not apparent, high-intensity 273 bp bands are evident for Lanes 2-4; however, this technique cannot be considered quantitative. A false positive signal in the band for ASP-PCR was also noted. C) The band intensity was almost same for 0.5%, 99.5%, and 100% samples. This method was considered to be unsuitable for quantitation. D) The template concentration of edited plasmid was considered to be important for this assay condition. A minimum presence of original edited plasmid in the amount of between 0.001 ng and 0.0001 ng is regarded as a lower limit for practical measurement. ASP, allele-specific primers.

PCR using allele-specific primers (ASP)
So we could ascertain the detection limit, in standard dilutions, the PCR products of edited and nonedited plamids were mixed in differing proportions. When we examined the results of these trials, it was apparent that it was better for the edited site to be situated in the forward primer. If sited in the reverse primer, the 3' end would be G and this would greatly increase the likelihood of nonspecific amplification. After designing a pair of allele-specific primers ( Fig. 1 and 2A), F>5'AAGGACTCACTTTGTTT3' and R>GGGATCCAGAACTCT3', we carried out PCR using allele-specific primers with rTth DNA Polymerase XL (Applied Biosystems, Tokyo, Japan). PCR conditions were 25 cycles of 2 min at 94°C, 15 s at 94°C and 30 s at 60°C, another 40 cycles of 15 s at 94°C, and 30 s at 57°C.
After PCR using allele-specific primers, the PCR products were electrophoresed on 1% agarose gel at 100 V/cm in 1×TAE. Following electrophoresis, gels were incubated in SYBER Green solution for 1 min. Gels were scanned directly at 488 mm using a Fluor-oImager (Molecular Dynamics, Sunnyvale, CA). SYBR Green fluorescence was captured using a 530DF30 filter with the photomultiplier tube voltage was set at 700 V.
Scans were analyzed using ImageQuant software (Amersham Bioscience, Tokyo, Japan). Band intensities were compared using the average value of the integrated pixel intensity for a two-pixel line drawn through the middle of each lane. We analyzed the band intensity by calculating when band intensity for edited plasmids would be 100% ( The value of the each band intensity(%) = the actual value of each sample intensity / the actual value of 100% edited plasmid's band intensity *100) . When we analyzed the detection limit, the band intensity of the 0.1 ng template was estimated to have a value of 100. Results for the different samples were indexed on this scale of 100.

DHPLC analysis
Denaturing HPLC was performed using a WAVE DNA fragment analysis system with a DNASep column (Transgenomic, Tokyo, Japan). To generate heteroduplex molecules, PCR products were subjected to denaturation for 4 min at 94°C followed by a gradual reannealing for 45 min in the thermal cycler. Aliquots of 5 μl of reannealed PCR products were loaded in a column. The gradient was prepared by mixing TEAA buffer A (0.1 M triethylammonium acetate). Primer extension products were eluted using a linear gradient from 18%B to 38%B at a flow rate of 0.9 ml/min for 7 min. Column temperature was set at 57.9°C. The eluted products were monitored at 260 nm using the UV detector. After each elution, the column was washed and equilibrated with the gradient of 90%B and 18%B, each for 1 min. Peaks were identified using standard PCR products as described above. RNA editing efficiency was calculated by comparing the peak heights after peak fitting with Gaussian curves corresponding to edited and non-edited extension products. The RNA editing efficiency of each sample was analyzed in duplicate.

Direct sequencing
Standard PCR products were purified using column purification on glass fibers (Qiagen ,Tokyo, Japan). We mixed the two purified standard PCR products at various ratios. These mixed samples were sequenced on an ABI PRISM3100 Genetic Analyzer using BigDye terminator v1.1 sequencing kits (both Applied Biosystems, Tokyo, Japan). The raw relative peak amplitude data for each sample was analyzed.
To derive the editing ratio of the samples, we designed two TaqMan® MGB probes for quantitation using FAM and VIC, whose sequences matched edited and nonedited sites (GlyR alpha3 nonediting probe: TCACTTTGCCTCAATT and GlyR alpha3 editing probe: ACTCACTTTGCTTCAAT)) and a pair of flanking primers (GlyR  alpha3  1017F,  AGCACCAGTACAAGTGGCTGAA; and 1092R, GCAGTATCGCAAATCTTTTTCTTCT). Next, we mixed samples with edited and nonedited plasmids included at various known ratios. Each allele-specific real-time PCR reaction mixture contained a 5 μl mixed plasmid sample, 4.5 μl (900 nM) of 10 pmol/μl forward primer, 4.5 μl (900 nM) of 10 pmol/μl reverse primer, 1.0 μl (200 nM) of 10 pmol/μl TaqMan editing probe, 1.0 μl (200 nM) of 10 pmol/μl TaqMan nonediting probe, 25 μl of TaqMan Universal PCR Master Mix. Total reaction volume was 50 μl. The amplification program consisted of 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 s denaturation at 95°C, and annealing for 1 min at 60°C. The quantities of each allele were determined using the comparative Ct method with these two probes and analysis on a real time PCR system (Applied Biosystems 7300; Tokyo, Japan). Regression analysis was performed on the results for samples of each ratio.

Statistical analysis
The correlation coefficient was obtained using simple regression analysis (Microsoft Excel; Redmond, WA).

Results
Our goal was to find and compare inexpensive assays that gave accurate quantitative or sensitive results. We found one method that was solely sensitive and three that were sensitive and quantitative.

Detection of RNA editing using allele-specific primers
Using allele-specific primers, we were able to detect a 0.5% incidence of editing sites. Figure 2 shows results using allele-specific primer (ASP) assays with control reactions. When the incidence of edited transcripts was more than 0.5%, 267 bp bands were clearly visible when the samples were analyzed (Fig. 2B). Intensity in these bands was not the same as for non-edited plasmid samples and we concluded that this method, while not quantitative, did enable detection and that the detection limit was between 0.001 ng and 0.0001 ng. (Fig. 2C, 2D)

Detection and quantitation of RNA editing by direct sequencing
To perform determine the accuracy of this assay, we mixed edited and non-edited GlyR alpha 3 standard PCR products (Fig. 3). The two kinds of PCR product, otherwise identical, contained either a C or a T at position 554. From the raw direct-sequencing data, peak intensities of fluorescence for each of the variously edited and nonedited samples were recorded. Fitting the data with a linear R2 value of 0.986, these peaks accurately reflected the ratios in the plasmid mixes. Detection was only sensitive down to an incidence of 5% of the total, lower incidences of editing were not detected (Fig. 3).

Detection and quantitation of RNA editing using DHPLC analysis
DHPLC (denaturing high performance liquid chromatography) is commonly used for detecting mutations through screening of PCR products for heterogeneity (Fig. 4). While edited products could be detected even when they made up as little as 2% of the total transcripts (Fig. 4 above), at levels below 10%, it was difficult to analyze DHPLC data to accurately determine actual quantities. We also tested mixtures that contained different percentages of nonedited and edited PCR products and Figure 4 shows the results for this DHPLC. For samples containing 10% to 90% mutant transcripts, these results accurately reflect the plasmid-mix ratios, fitting the data with a linear R 2 value of 0.972.

Implementation of allele-specific real-time PCR method with TaqMan probe
We confirmed that the amplification efficiencies of primers used for edited and nonedited samples were approximately equal (data not shown). We tested two MGB probes at various ratios of control plasmid and confirmed that they were specific to each clone of Gly alpha3 (Fig.  5B). In other words, edited probes were highly specific only for edited transcripts and the nonedited probed were highly specific only for nonedited transcripts. Then, we analyzed various ratios of edited and nonedited products using these primers and probes. We were able to determine the editing ratios at y = 25.237 e -0.9337X (R 2 = 0.9919; y, measured value; x, actual value) (Fig. 5C). Each chromatogram shows results of DHPLC analysis for PCR products amplified using mixed templates of different ratios of editing (0%-5%, 95%-100%). Although two distinguishable heteroduplex and homoduplex peaks were not apparent, a small peak (heteroduplex) next to a large peak was seen in mixes in which 2% or more of plasmids were edited. Quantitation of known template assay results. The least-squares fit of the actual data is defined by the equation y = 0.718x + 13.1, p = < 0.001. Figure 5. (A) Two TaqMan ® MGB probes using FAM TM and VIC ® , whose sequences matched edited or nonedited sites, and a pair of flanking primers was designed to increase the precision of allele-specific quantitation. In the sequence, C/T denotes the edited site. (B) The quantities of each allele were evaluated from ∆Ct results from a real-time PCR system (TaqMan ABI 7300). (C) Quantitation using allele-specific real time PCR methods. Assuming that ∆Ct was equal to zero when the editing ratio was 50%, logistic regression analysis of the incidence of editing in differently mixed samples was performed. Correlation between measured values and corrected ∆Ct is shown. The actual data is defined by the equation y = 25.237e -0.9337x , p = < 0.001 (y: measured value, x: actual value).

Discussion
The ability to measure subtle differences in editing efficiency is becoming increasingly important, particularly for clinical studies. For example, minute reductions in editing efficiency have been associated with clinical depression, suicide, certain forms of epilepsy, and schizophrenia [4,17,18,19,20,]. The properties of alpha amino-3-hydroxy-5-methyl-4-isoxazoleprpionate (AMPA) receptor are generated post-transcriptionally by RNA editing at the Q/R site, where deficient RNA editing of GluR2 is associated with neuronal death in amytropic lateral sclerosis [21,22]. Recently, a number computational methods for detecting A-to-I RNA editing sites have been proposed [23], and algorithmic approaches for identifying RNA editing sites have been discussed [24]. The methods of testing that we have demonstrated here are useful, at the study planning stage, for establishing the means of detecting and quantifying editing ratios within a short time.
We found that the best way of detecting of already known RNA editing was PCR with allele-specific primer pairs. If good primer pairs are created, this method is simple and inexpensive; however, because the polymerase amplification is susceptible to approximately 1% error [25], it is not always accurate. The relevant analytical efficacy of this low-cost option must be carefully considered. Our PCR results with ASPs were excellent and useful for identifying if editing had occurred.
If it is not possible to properly design ASPs, DHPLC may be feasible. The DHPLC method has been used successfully for detecting point mutations at very low (0.5%-5.0%) percentages of heterogeneity [26,27]. DHPLC may be useful because it can inexpensively provide analysis of known editing. Using direct sequencing, assay detection of editing incidences is 20% in general, and in our analysis we managed to detect, and measure accurately, incidences as low as 5%. Unfortunately, direct sequencing incurs high costs. Thus, direct sequencing is too expensive to use from the beginning. Another option, if the editing site is already known, is the use of allele-specific TaqMan probes. If the primer and probe are designed especially for detection, this method can provide detectability down to 2% (data not shown). But the primer-probe pair which we designed for detection was not at all useful for quantitation. Furthermore, there is no special cost benefit in using allele-specific TaqMan probes.
For quantitating already known RNA editing ratios, direct-sequencing and DHPLC used in combination with allele-specific TaqMan probes were useful.
Compared against the edited/nonedited ratios of known standard samples, results for DHPLC (R 2 = 0.973), from allele-specific TaqMan probes (R 2 = 0.992), and from direct sequencing (R 2 = 0.987) correlated well. Allele-specific TaqMan probes yielded the closest correlation because they were designed and used in conditions that favored quantitation of the incidence of editing. Conversely, such probes are not suitable for blind detection. Even so, once the primers and probe are designed and evaluated, this method is both accurate and easy to perform. Consequently, it is very useful for evaluating samples in which RNA editing is known to have occurred. Meanwhile, DHPLC is inexpensive and has good detection sensitivity down to 2%. So DHPLC would appear to be an excellent means both of RNA editing detection and quantitation. Our results using DHPLC, however, did manifest a shortcoming. In the protocol we used, we were unable to determine four peaks, which should be detectable in ideal conditions. We were unable to ascertain which peaks were edited or non-edited. For confirmation, supplementary procedures involving standard samples were necessary. Of the methods we used for detecting and quantitating samples in which partial editing at the site of GlyR alpha3 was present, we conclude that ASP was best for detection and that TaqMan probes or direct sequencing gave superior quantitation results.
Recently, computational methods for detecting A-to-I RNA editing sites have been introduced [23]. Such analysis has revealed thousands of editing sites within the genomic reiteration, and has also brought to light some novel genetic recoding sites. If computational methods are used for prediction, preliminary testing of predicted gene clones could be simply and inexpensively carried out using ASP. DHPLC could then be used to review non-conforming ASP results. Furthermore, when it is known that editing has occurred in a real sample, direct sequencing can be used to confirm the suspected editing site. When priority is given to quantitation, TaqMan probes or direct sequencing should be used.
For detecting and quantitating a specific type of editing, ASP and DHPLC are suitable for initial detection, followed by quantitation using direct sequencing or allele-specific TaqMan probes. To screen for unknown editing, from the options tested, DHPLC or direct sequencing should be considered.
It is, of course, essential to carry out validation using clones of each sample. We suppose that for any type of unknown RNA editing, the techniques reported here can enable identification and quantitation of the SNP. It is important, however, to use the method that is most appropriate for the specific pur-pose and circumstance.
In conclusion, we evaluated four different ways of detecting and quantitating the incidence of RNA editing. These techniques and our findings should be of interest to anyone carrying out research on other RNA editing sites, especially single-base editing patterns. The methodologies presented here are likely to be beneficial for current and future studies aimed at understanding the relationship between the editing incidence of a target gene and the effectiveness of corresponding physiological or pharmacological phenotypes.


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A quantitative study of spinothalamic neurons in laminae I, III, and IV in lumbar and cervical segments of the rat spinal cord

The major ascending outputs from superficial spinal dorsal horn consist of projection neurons in lamina I, together with neurons in laminae III–IV that express the neurokinin 1 receptor (NK1r) and have dendrites that enter the superficial laminae. Some neurons in each of these populations belong to the spinothalamic tract, which conveys nociceptive information via the thalamus to cortical areas involved in pain. A projection from the cervical superficial dorsal horn to the posterior triangular nucleus (PoT) has recently been identified. PoT is at the caudal end of the thalamus and was not included in injection sites in many previous retrograde tracing studies. We have injected various tracers (cholera toxin B subunit, Fluoro-Gold, and fluorescent latex microspheres) into the thalamus to estimate the number of spinothalamic neurons in each of these two populations, and to investigate their projection targets. Most lamina I and lamina III/IV NK1r-immunoreactive spinothalamic neurons in cervical and lumbar segments could be labeled from injections centered on PoT. Our results suggest that there are 90 lamina I spinothalamic neurons per side in C7 and 15 in L4 and that some of those in C7 only project to PoT. We found that 85% of the lamina III/IV NK1r-immunoreactive neurons in C6 and 17% of those in L5 belong to the spinothalamic tract, and these apparently project exclusively to the caudal thalamus, including PoT. Because PoT projects to second somatosensory and insular cortices, our results suggest that these are major targets for information conveyed by both these populations of spinothalamic neurons.

The spinal dorsal horn receives a major input from primary afferent axons. This input is organized according to modality, with nociceptive afferents terminating mainly in laminae I and II (Rexed, 1954;Todd and Koerber, 2005). Although the axons of most dorsal horn neurons remain within the spinal cord, some of these cells (projection neurons) have axons that terminate in the brain, and these form an important output from the region. Projection neurons are not uniformly distributed within the dorsal horn, but are concentrated in lamina I and scattered throughout the deeper laminae (III-VI).
The majority of lamina I projection neurons in the rat express the neurokinin 1 receptor (NK1r) on which substance P acts (Ding et al., 1995;Li et al., 1996Li et al., , 1998Marshall et al., 1996;Todd et al., 2000;Spike et al., 2003). Another population of projection neurons consists of large NK1r-immunoreactive cells with somata in laminae III or IV and long dorsal dendrites that reach lamina I (Naim et al., 1997;Todd et al., 2000). Ablation of NK1r-expressing neurons in the superficial dorsal horn results in loss of hyperalgesia in chronic pain models (Mantyh et al., 1997;Nichols et al., 1999), which suggests that projection neurons with the NK1r may play an important role in the development of hyperalgesia. The supraspinal projection targets of these two groups of cells have been studied most extensively in the rat and include the caudal ventrolateral medulla (CVLM), the lateral parabrachial area (LPb), the periaqueductal gray matter (PAG), and the thalamus (Menétrey et al., 1982(Menétrey et al., , 1983Cechetto et al., 1985;Hylden et al., 1989;Lima and Coimbra, 1988;Burstein et al., 1990;Lima et al., 1991;Ding et al., 1995;Todd et al., 2000;Spike et al., 2003;Gauriau and Bernard, 2004a).
These projections are predominantly contralateral, although a significant number of cells appear to project bilaterally (Spike et al., 2003). Both lamina I and lamina III-IV projection neurons that possess the NK1r are densely innervated by substance P-containing primary afferent axons (Naim et al., 1997;Todd et al., 2002) and thus provide a route through which these afferents, which are known to be nociceptors (Lawson et al., 1997), can activate brain regions involved in pain mechanisms.
It has been shown that some of the lamina III/IV NK1rimmunoreactive neurons in rat lumbar spinal cord project to the thalamus (Marshall et al., 1996;Naim et al., 1997). However, the proportion of those in lumbar or cervical cord that belong to the spinothalamic tract is not known.
In the present study we have re-examined the spinothalamic projection from lamina I and from the NK1rexpressing lamina III/IV cells of lumbar and cervical segments of the rat spinal cord. Our main aim was to address the following questions: 1) has the number of spinothalamic neurons in lamina I been underestimated in previous studies, due to failure to fill the PoT?; 2) is there a projection from lumbar segments to PoT?; and 3) what proportion of lamina III/IV NK1r-expressing cells in lumbar and cervical cord belong to the spinothalamic tract and where do their axons terminate within the thalamus?
A recent study by Yu et al. (2005) reported that spinothalamic neurons made up 9% of the neuronal population in lamina I of the rat cervical cord. However, we have provided evidence that only ϳ5% of lamina I neurons in lumbar cord project to the brain (Spike et al., 2003), and it has been shown that in the cat, spinothalamic lamina I cells are greatly outnumbered by other projection neurons (Mouton and Holstege, 1998;Klop et al., 2005b). Therefore an additional aim of this study was to determine the proportion of lamina I cells in cervical cord that belong to the spinothalamic tract.

MATERIALS AND METHODS Surgery
All experiments were approved by the Ethical Review Process Applications Panel of the University of Glasgow and were performed in accordance with the UK Animals (Scientific Procedures) Act 1986.
Twenty-five adult male Wistar rats (240 -330 g; Harlan, Loughborough, UK) were anesthetized either with ketamine and xylazine (73.3 and 7.3 mg/kg i.p., respectively, supplemented as necessary) or with a volatile anesthetic (halothane or isofluorane). They were then placed in a stereotaxic frame and in those cases in which halothane or isofluorane were used, the anesthetic was administered through a mask attached to the frame. Each rat received injections of one or more of the following tracers into the left thalamus: 1% cholera toxin B subunit (CTb; Sigma, Poole, UK), 4% Fluoro-Gold (Fluorochrome, Englewood, CO), or rhodamine-labeled fluorescent latex microspheres (undiluted, Lumafluor, Naples, FL). All injections were made through glass micropipettes, and these were left in place for 5 minutes after the completion of each injection to minimize leakage of tracer back up the track. In those cases in which two tracers were injected, a different pipette was used for each tracer. Four different injection strategies were used (Table 1) Details of the tracer(s) used and volumes injected in each experiment are given in Table 1. The experiments with fluorescent latex microspheres were performed because these show very little spread (resulting in restricted injection sites) and are not taken up by undamaged fibers (Katz et al., 1984).
All animals made an uneventful recovery from anesthesia. After a survival period of 3 days they were reanesthetized with pentobarbitone (300 mg i.p.) and perfused through the heart with a fixative that contained 4% freshly depolymerized formaldehyde. Lumbar and cervical spinal cord segments were removed and stored in fixative for 24 hours, whereas the brain was cryoprotected in 30% sucrose in fixative overnight. In order to measure the length of the C7 segment, this was photographed in situ in three experiments (DI5 and 6 and FLM3), and the distance between the most rostral and caudal rootlets of the C7 dorsal roots was determined.

Tissue processing and immunocytochemistry
The brain region surrounding the injection site was cut into 100-m-thick coronal sections with a freezing microtome. Brains from animals that had received injections of CTb (with or without Fluoro-Gold) were cut into five complete series, and sections from at least one of these series were reacted with goat anti-CTb (List Biological Laboratories, Campbell, CA; diluted 1:50,000) by using an immunoperoxidase method as described previously (Spike et al., 2003). All remaining sections from animals that had received injections of Fluoro-Gold in addition to CTb were mounted with anti-fade medium (Vectashield, Vector, Peterborough, UK) for detection of Fluoro-Gold with epifluorescent illumination and an UV filter set. All sections through the injection sites from animals that had received only Fluoro-Gold or fluorescent latex microspheres were mounted in serial order. Those from Fluoro-Gold-injected rats were mounted with Vectashield and viewed with UV epifluorescence. Sections from animals that received injections of fluorescent latex microspheres were mounted in Gel-Mount medium (Sigma-Aldrich, Poole, Dorset, UK) because the microspheres are destroyed by glycerol, and these were viewed with bright-and darkfield illumination. In all cases the spread of tracer from the injection sites was plotted onto drawings of the thalamus (Paxinos and Watson, 2005). Representative examples were photographed.
The C7 and L4 spinal cord segments from all animals, apart from those that received injections of fluorescent latex microspheres, were initially notched on the left side, so that the two sides could subsequently be distinguished, and were then cut into 60-m-thick transverse sections with a Vibratome. Some or all of these sections were reacted to reveal neuronal nuclei, the tracer(s), and in some cases the NK1r. Sections were incubated freefloating at 4°C for 3 days in one of the following antibody combinations: 1. Mouse monoclonal antibody NeuN (against a neuronal nuclear protein, Millipore, Watford, UK; diluted 1:1,000), goat anti-CTb (1:5,000), and rabbit anti-NK1r (Sigma-Aldrich; 1:10,000) (experiments PoT1 and PoT7-10).
The sections were then incubated overnight at 4°C in species-specific secondary antibodies that were raised in donkey and conjugated to either Alexa 488 (Invitrogen, Paisley, UK; 1:500), or to Rhodamine Red or Cy5 (Jackson ImmunoResearch, West Grove, PA; 1:100). Sections from experiments Thal1 and PoT2-3 were incubated for 3 days in rabbit anti-Fluoro-Gold (Millipore, diluted 1:5,000), overnight in the FabЈ fragment of goat anti-rabbit IgG conjugated to rhodamine (Jackson ImmunoResearch), and then for 2 days in rabbit anti-NK1r, followed by 1 day in donkey anti-rabbit IgG conjugated to Alexa 488. Following immunostaining, some of the sections from the C7 segment of experiments Thal2-5 were incubated for 30 minutes in the fluorescent nuclear stain 4Ј,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich; 1 g/ml in phosphatebuffered saline). All sections were mounted in Vectashield and stored at Ϫ20°C.
The sections were then incubated overnight at 4°C in species-specific secondary antibodies that were raised in donkey and conjugated to Alexa 488, Rhodamine Red, or Cy5. Sections from experiments Thal1 and PoT2-3 were processed sequentially in rabbit antibodies against Fluoro-Gold and NK1r, as described above. Sections were mounted in serial order in Vectashield or Gel-Mount and stored at Ϫ20°C.

Antibody characterization
The NK1r antibody (catalogue number S8305) was raised in rabbit against a peptide corresponding to amino acids 393-407 at the C-terminus of the rat NK1 receptor, which was conjugated to keyhole limpet hemocyanin. The antibody recognizes a 46-kDa band in Western blots of rat brain extracts, and this staining is specifically abolished by preabsorption of the antibody with the immunizing peptide (manufacturer's specification). It has been shown that there is no immunostaining with this antibody in sections of medulla and cervical spinal cord from mice in which the NK1r has been deleted (NK1Ϫ/Ϫ), whereas staining is present in sections from wild-type mice (Ptak et al., 2002).
The mouse monoclonal antibody NeuN (catalogue number MAB377) was generated against cell nuclei extracted from mouse brain and was found to react with a protein specific to neurons (Mullen et al., 1992). We have shown that this antibody apparently labels all neurons (and no glial cells) within the rat spinal cord . The goat polyclonal antibody (catalogue number 703) was raised against CTb, whereas rabbit (catalogue number AB153) and guinea-pig (catalogue number NM101) antibodies were raised against Fluoro-Gold. Specificity of each of these tracer antibodies was shown by the lack of staining in regions of the CNS that did not contain neurons that had taken up and transported the tracer and by the presence of immunostaining in populations of neurons that are known to project to the injection sites. In addition, the specificity of the Fluoro-Gold antibodies was directly confirmed by comparing Fluoro-Gold fluorescence (observed with an UV filter set) with that for anti-Fluoro-Gold in individual neurons. In all cases examined, there was a perfect match between the two types of fluorescence.

Confocal microscopy and analysis
Transverse sections from the rats that had received injections of CTb and/or Fluoro-Gold were used to analyze the number of retrogradely labeled neurons in lamina I in each experiment. From each animal, 10 sections from the C7 segment and 20 sections from the L4 segment were randomly selected and viewed with epifluorescence. All lamina I neurons on the right side of each section that were labeled with CTb and/or Fluoro-Gold were identified and then scanned with a confocal microscope (Bio-Rad MRC1024 or Radiance 2100; Bio-Rad, Hemel Hempstead, UK) through dry (4ϫ, 10ϫ, 20ϫ) and oil-immersion (60ϫ) lenses. In order to correct for the overcounting that results from the presence of transected cells at the section surfaces, cells were only included in the sample if their nucleus (identified as a filling defect) was entirely contained within the Vibratome section, or if part of the nucleus was present in the first optical section in the z-series (corresponding to the top of the Vibratome section); they were excluded if part of the nucleus was present in the last optical section in the z-series (Spike et al., 2003). The low-magnification images were used to plot the position of the cells on an outline of the dorsal horn. In this way the mean number of retrogradely labeled lamina I cells containing CTb, Fluoro-Gold, or both tracers per 600 m (C7) or 1,200 m (L4) was determined for each experiment. Darkfield microscopy was used to distinguish laminar boundaries (see Results), and retrogradely labeled cells were judged to be in lamina I if they were very close to the dorsal border of the dorsal horn or lay dorsal to the dark band identified as lamina II with darkfield microscopy. For most of the experiments in which a single tracer was used (Thal2-5, PoT1, PoT4 -10), the presence or absence of NK1r was also noted for each retrogradely labeled lamina I cell.
In order to determine the proportion of lamina I neurons that belonged to the spinothalamic tract in the C7 segment, six Vibratome sections that had been immunostained and incubated in DAPI were randomly selected from four experiments in which extensive injections of Fluoro-Gold had been made (Thal2-5). These were scanned through a 40ϫ oil-immersion lens to reveal NeuN, NK1r, Fluoro-Gold, and DAPI. In each case, z-series consisting of 16 optical sections at 1 m spacing were obtained from the entire mediolateral extent of lamina I on the right side of the spinal cord. To determine the degree of tissue shrinkage, the thickness of each section was measured by scanning top and bottom surfaces at three different locations and calculating the mean distance between these surfaces (Polgá r et al., 2004). Drawings of these sections were made with Neurolucida for Confocal (MicroBrightField, Colchester, VT), and the boundaries of lamina I were identified, based on the pattern of NK1r immunostaining (see Results). A modification of the optical disector method (Sterio, 1984) was then used to determine the total number of neurons in a 9-mthick slice through lamina I, by examining all optical sections and counting all of the neurons with nuclei that had a bottom surface between the 3rd and 11th optical sections in the z-series Spike et al., 2003). To avoid bias, the selection of neurons was made before the Fluoro-Gold immunoreactivity was viewed. For each neuron that was included in the disector sample, the presence or absence of Fluoro-Gold was then noted. The proportion of lamina I neurons that belonged to the spinothalamic tract was estimated in two different ways.
First, for each rat the number of retrogradely labeled neurons in the disector samples was divided by the total number of lamina I neurons in the samples, and this value was averaged for the four rats. Second, the disector samples were used to estimate the total number of lamina I neurons per 600 m in the C7 segment, and this was subsequently compared with the numbers of retrogradely labeled lamina I neurons found in this segment in the experiments in which CTb and/or Fluoro-Gold were injected. In order to estimate the total number of lamina I neurons in this way, we used the following formula to correct for tissue shrinkage (Polgá r et al., 2004): where N is the number of lamina I neurons per 600 m of spinal cord, n is the number counted in the 9-m disector, T-final is the thickness of the particular section measured after any shrinkage, and T-cut is the original section thickness cut (60 m).
For each of the rats that had received injections of CTb and/or Fluoro-Gold, the entire series of parasagittal sections through the gray matter of the right side of both the C6 and L5 segments were used to count the number of NK1 receptor-immunoreactive neurons that had cell bodies in laminae III or IV and dorsal dendrites that could be followed into laminae I or II, and to determine the proportion of these cells that was retrogradely labeled. Darkfield microscopy was used to ensure that all cells had their somata ventral to lamina II. Sections were initially viewed with epifluorescence through a 20ϫ lens to identify NK1 receptor-immunoreactive cell bodies in laminae III or IV. In most cases it was possible to determine with epifluorescence whether the dendrites of these cells entered the superficial dorsal horn (laminae I or II). However, in some cases (particularly when dendrites had to be followed into serial sections) it was necessary to scan with the confocal microscope. Confocal microscopy was also used to determine whether the cells were retrogradely labeled with CTb and/or Fluoro-Gold, to measure the distance between the cell body and the overlying dorsal white matter, and to ensure that cell bodies that were cut by the Vibratome and appeared on the adjacent surfaces of serial sections were The Journal of Comparative Neurology not counted twice. During the course of the study (see Results), we observed that retrogradely labeled lamina III/IV NK1r-immunoreactive neurons in the L5 segment appeared to be more common in the medial part of the dorsal horn than in the lateral part. We investigated this by assigning all of the NK1r-immunoreactive cells in this segment with dorsal dendrites that entered the superficial dorsal horn into one of two groups, medial and lateral, based on the section in which they were present and, if necessary, their (mediolateral) depth within that section. In experiments in which the number of these cells was even, the two groups were of equal size, whereas in the other experiments the extra cell was included in the lateral group.
For the three rats that had received injections of fluorescent latex microspheres (FLM1-3), complete series of parasagittal sections through the right side of the C6 and L5 segments were examined with epifluorescence through a 40ϫ oil-immersion lens. All retrogradely labeled neurons in lamina I, as well as retrogradely labeled neurons in laminae III and IV that were NK1r-immunoreactive, were identified. Care was taken to avoid double-counting by ensuring that cells at the cut surface of a section were not counted on the adjacent section. For the retrogradely labeled lamina III/IV cells we determined whether dorsal dendrites could be followed into the superficial laminae (I-II) in each case.
Figures were composed with Adobe Photoshop (version 7.0). In some cases, image brightness and contrast were adjusted by using the levels setting.

Injection sites for CTb and Fluoro-Gold experiments
The spread of tracer within the thalamus in each of these experiments is illustrated in Figures 1-3, and representative photomicrographs of injection sites are shown in Figure 4.
Injections of Fluoro-Gold aimed at thalamic regions known to receive projections from the superficial dorsal horn (Thal1-5) were successful in all cases (Fig. 1). The injection sites included PoT, the posterior thalamic nuclear group (Po), and the ventrobasal (VB) complex (ventral posteromedial nucleus, ventral posterolateral nucleus [VPL], and ventral posterior nucleus parvicellular part). In experiments Thal2-5 there was also variable filling of the medial thalamus. There was some spread of tracer into regions surrounding PoT (e.g., medial geniculate nucleus [MG] and anterior pretectal nucleus [APT]) in each experiment, together with variable extension into the basal ganglia. There was no spread of tracer into the hypothalamus.
Injections targeted on the PoT (Fig. 2) filled all or most of this region in seven experiments (PoT3-9), whereas tracer filled only the rostral half in PoT1 and the caudal part in PoT2; the injection in PoT10 was too far rostral and involved mainly the caudal part of the Po. There was invariably spread into nearby structures such as: Po, MG, and APT, as well as suprageniculate, lateral posterior, and posterior intralaminar (PIL) thalamic nuclei. In experiment PoT2 there was significant spread of tracer into the midbrain but not into the PAG. In several cases (PoT1, 3-6, and 10) there was some rostral spread of tracer into the VB complex. There was no spread of tracer into the hypothalamus in any of these experiments.
In the double injection experiments, Fluoro-Gold was targeted on thalamic nuclei rostral to interaural 4.0 mm, and these were partially (DI1-4) or almost completely (DI5-7) filled (Fig. 3). Fluoro-Gold extended caudally to involve rostral PoT in DI4, and into caudal Po in DI1, 3, and 7. PoT was partially or completely filled with CTb in all experiments. Neither tracer spread into the hypothalamus in any of these experiments.

Quantitative analysis of lamina I spinothalamic tract neurons in C7 and L4
The width of lamina I in the C7 segment (as judged by darkfield microscopy) was somewhat greater in the central region (up to 35 m) than in the lateral or medial parts, where it was generally between 10 and 15 m thick (Fig. 5a). An identical pattern was seen with the plexus of NK1r-immunoreactive dendrites and cell bodies that occupies lamina I (Fig. 5b).
The distributions of retrogradely labeled cells were generally similar across all of the experiments in which CTb or Fluoro-Gold were used as tracers. In the C7 segment, moderate numbers of retrogradely labeled neurons were observed in each section, and these were found in lamina I and scattered throughout the deeper laminae (III-VI) of the dorsal horn and the ventral horn. Occasional labeled cells were seen in lamina II. Although the distribution of retrogradely labeled cells in laminae I-V was similar in the L4 segment, these cells were far less numerous, for example, most transverse 60-mVibratome sections contained no labeled lamina I neurons.
Quantitative data for retrogradely labeled lamina I neurons are shown in Tables 2-4. The locations of retrogradely labeled cells in the upper part of the dorsal horn that were sampled in a representative experiment are illustrated in Figure 6, and examples of labeled lamina I cells are shown in Figure 7. The mean number of lamina I cells per 600 m in the C7 segment was 23.2 for experiments Thal1-5 and 24.6 for experiments PoT1-9. (Data from PoT10 were not included in this part of the analysis, because the injection did not spread into PoT.) The corresponding numbers per 600 m for the L4 segment were 3.5 for Thal1-5 and 3.6 for PoT1-9 (Tables 2, 3). In experiments DI1-7, the mean numbers of cells that were retrogradely labeled (with either tracer) per 600 m in the C7 and L4 segments were 22.4 and 4.6, respectively (Table 4). Comparison of these values with those obtained in experiments Thal1-5 and PoT1-9 indicated that there were no significant differences between the numbers of retrogradely labeled cells found in each series for either cervical or lumbar segments (one-way ANOVA, P Ͼ 0.05 for C7 and L4).
In all of the double injection experiments apart from DI6, the majority of retrogradely labeled lamina I cells in the C7 segment contained CTb. In the L4 segment the majority of labeled lamina I cells contained CTb except in experiments DI6 and 7 (Table 4).
For experiments Thal 2-5, PoT1, and PoT4-10, the retrogradely labeled lamina I neurons were examined for the presence of NK1r immunoreactivity. When data from these experiments were pooled, it was found that 255 out of 294 (87%) of the cells in C7 and 65 out of 77 (84%) of those in L4 were NK1r immunoreactive (Fig. 8).
In the sections of C7 that were used to determine the proportion of lamina I neurons belonging to the spinothalamic tract, numerous neuronal (NeuN-positive) profiles with DAPI-labeled nuclei were visible in lamina I, and The Journal of Comparative Neurology  Paxinos and Watson (2005). AM, anteromedial thalamic nucleus; APT, anterior pretectal nucleus; AV, anteroventral thalamic nucleus; CL, centrolateral thalamic nucleus; CM, central medial thalamic nucleus; fr, fasciculus retroflexus; IAM, interanteromedial thalamic nucleus; ic, internal capsule; LD, laterodorsal thalamic nucleus; LG, lateral geniculate nucleus; LP, lateral posterior thalamic nucleus; MD, mediodorsal thalamic nucleus; MG, medial geniculate nucleus; PC, paracentral thalamic nucleus; PF, parafascicular thalamic nucleus; PIL, posterior intralaminar thalamic nucleus; Po, posterior thalamic nuclear group; PoT, posterior thalamic nuclear group, triangular part; PP, peripeduncular nucleus; Re, reuniens thalamic nucleus; RRE, retrouniens area; Rt, reticular thalamic nucleus; Sub, submedius thalamic nucleus; SubB, subbrachial nucleus; VA, ventral anterior thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; VPPC, ventral posterior thalamic nucleus, parvicellular part. Scale bar ϭ 1 mm (also applies to Figs. 2 and 3). many of these were also NK1r-immunoreactive (Fig. 8). Although Fluoro-Gold-positive cells were seen in lamina I in most sections, these were greatly outnumbered by neurons that were not retrogradely labeled. The total numbers of lamina I neurons in the disector samples in each experiment varied from 139 to 168 (mean 150, n ϭ 4), whereas the numbers that were retrogradely labeled in these samples ranged from 1 to 7 (mean 3.5). The mean percentage of the sampled lamina I neurons that were retrogradely labeled was 2.4% (range 0.6 -5%). Estimates of the total number of lamina I neurons per 600 m length of C7 (calculated according to the formula in Materials and Methods) for the four experiments ranged from 1,028 to 1,420 (mean 1,254). The mean number of retrogradely labeled lamina I neurons determined for all PoT, Thal, and DI experiments (excluding PoT10; see above) was 23.5 per 600 m (data from Tables 2-4), and this corresponds to 1.9% of the estimated total population of lamina I neurons.

Proportion of lamina III/IV NK1rimmunoreactive neurons that project to thalamus
As reported previously (Brown et al., 1995;Littlewood et al., 1995) we observed large NK1r-immunoreactive neurons in laminae III and IV in parasagittal sections of the L5 segment, and these often had dendrites that could be followed into the superficial dorsal horn (laminae I-II) (Fig.  9b). Similar cells were observed in the C6 segment (Fig. 9a), although these were somewhat less numerous than those in L5 (Tables 5-7). When data from Thal, PoT, and DI experiments were pooled, the mean number of NK1rimmunoreactive cells with somata in laminae III-IV and dendrites that could be followed into the superficial dorsal horn was 22.1 on the right side in the L5 segment and 15.8 on the right side in C6. The cells were uniformly distributed along both the mediolateral and rostrocaudal extent of the dorsal horn in each segment. The cell bodies were located between 93 and 405 m (mean 223 m Ϯ 63, n ϭ 419 cells) below the dorsal white matter in L5 and between 82 and 349 In the C6 segment, most cells of this type were retrogradely labeled in all of the Thal, PoT, and DI experiments (except for PoT10; Tables 5-7). Comparison of the percentage values obtained in each group indicated that there were no significant differences between them (one-way ANOVA, P Ͼ 0.05). The overall proportion labeled was 85% (data  (Table 7).
In contrast, in the L5 segment the majority of the cells of this type were not retrogradely labeled in any of the Thal, PoT, or DI experiments (Tables 5-7). Comparison of the percentage values obtained in each group indicated that there were no significant differences between them (one-way ANOVA, P Ͼ 0.05). The overall proportion labeled was 17% (data pooled for all experiments apart from PoT10). In the DI experiments, all of the retrogradely labeled cells of this type in L5 contained CTb, and some of those in DI1, 4, and 6 were double-labeled (Table 7).
Results of the analysis of mediolateral distribution of retrogradely labeled lamina III/IV neurons are shown in Tables 5-7. When data from experiments Thal1-5,

The Journal of Comparative Neurology
PoT3-9, DI1-4, and DI6 -7 were pooled, the mean percentage of neurons in the medial group that were retrogradely labeled was 27, whereas for the lateral group the mean percentage was 7. These values were significantly different (P Ͻ 0.001, t-test). Figure 10 shows drawings of the spread of fluorescent latex microspheres in experiments FLM1-3 and an example of an injection site from FLM3. In all cases, the spread of tracer was very limited, compared with that seen with CTb or Fluoro-Gold.

Retrograde labeling with fluorescent latex microspheres
Examples of retrogradely labeled neurons are shown in Figures 11 and 12, and quantitative data are provided in Table 8. The numbers of microspheres in labeled neurons varied considerably: only cells that contained 5 or more were considered to be retrogradely labeled, and most such cells contained more than 10 microspheres. The optical properties of Gel-Mount were not as good as those of Vectashield, and although fluorescent latex microspherescould be identified through the depth of the section, it was difficult to follow NK1r-immunoreactive profiles deep within the sections. In the C6 segment, lamina I was found to contain between 15 and 46 retrogradely labeled cells, whereas 5-10 retrogradely labeled lamina III/IV NK1r-immunoreactive cells were observed, of which 1-6 had dendrites that could be followed into the superficial dorsal horn (Fig. 12a). Because of the difficulty in following NK1r-immunoreactive dendrites in these sections, only those cells that had dorsal dendrites that entered lamina II in the superficial part of the same section could be unequivocally identified as belonging to this type. In the L5 segment, no retrogradely labeled cells were detected in laminae I or III/IV in experiment FLM1, whereas in experiments FLM 2 and FLM3, 4 and 5 lamina I cells (respectively) and 4 and 6 lamina III/IV NK1r cells (respectively) were seen. Dorsal dendrites of two of the lamina III/IV NK1r-immunoreactive cells in experiment FLM3 could be followed into the superficial dorsal horn, and one of these is illustrated in Figure 12b. Although many of the retrogradely labeled lamina I cells in C6 and L5 could be identified as NK1r-immunoreactive (Fig. 11), when the cell was in the deepest part of the section, it was not possible to assess NK1r immunoreactivity, and therefore the proportion of cells that expressed the receptor was not quantified.

DISCUSSION
The major findings of this study are: 1) that injections of CTb or Fluoro-Gold targeted on PoT can label a high proportion of lamina I spinothalamic neurons at lumbar and cervical levels, 2) that ϳ17% of lamina III/IV NK1rimmunoreactive neurons with long dorsal dendrites in L5 and ϳ85% of those in C6 belong to the spinothalamic tract, and 3) that the lamina III/IV cells are not retrogradely labeled by injections of Fluoro-Gold restricted to the VB complex, rostral part of Po, and medial thalamus. The two thin lines indicate the dorsal and ventral borders of lamina II, which were determined from darkfield micrographs. The drawing on the left shows the cells seen in 10 randomly selected 60-m sections through the C7 segment, whereas the drawing on the right represents the cells in 20 such sections from the L4 segment. 1 Counts of retrogradely labeled lamina I cells in the experiments in which cholera toxin B (CTb) was targeted at PoT and Fluoro-Gold (FG) at other thalamic regions known to receive projections from superficial dorsal horn neurons. In each experiment, cells were counted in 10 randomly selected transverse 60-m sections from the C7 segment and in 20 such sections from the L4 segment. 2 These columns shows the percentage of all retrogradely labeled cells that contained CTb. Fig. 7. Retrograde labeling of lamina I spinothalamic tract neurons with CTb and Fluoro-Gold. The main part of the figure shows part of lamina I in a transverse section from the C7 segment of experiment DI3 immunostained to reveal CTb (green) and Fluoro-Gold (FG; magenta). A merged image is shown on the right. Several retrogradely labeled lamina I cells are present in this field. Two of these (arrows) contain both CTb and Fluoro-Gold, and others (two of which are indicated with arrowheads) are labeled with CTb but not Fluoro-Gold. The inset shows a single lamina I neuron in a transverse section from the L4 segment of experiment DI5 that was labeled with both CTb and Fluoro-Gold. The images were obtained from a projection of 19 (main part) or 18 (inset) confocal optical sections at 1-m z-spacing. Scale bar ϭ 20 m in far right panel (applies to all). Fig. 8. A lamina I spinothalamic neuron with NK1r. a: A transverse section through the medial part of lamina I in the C7 segment of experiment Thal3 scanned to reveal NeuN (red) and DAPI (blue). Neuronal nuclei contain both NeuN and DAPI and therefore appear magenta, whereas nonneuronal nuclei are blue. b: The same field scanned to reveal NK1r (green) and Fluoro-Gold (FG; magenta). The continuous and dashed lines show the upper and lower borders of lamina I, respectively. The arrow indicates a retrogradely labeled neuron in lamina I, which is also NK1r-immunoreactive. Two other NK1r-immunoreactive neurons that are not retrogradely labeled are shown with asterisks, and several neurons that are NK1r-negative and lack Fluoro-Gold are also present in lamina I. The upper inset shows DAPI staining in the nucleus of the spinothalamic neuron, and the lower inset shows the NK1r on its surface membrane. All images are projections of two optical sections at 1-m z-spacing. Scale bar ϭ 20 m in b (applies to a,b).

Thalamic projection targets of lamina I neurons
Retrograde (Granum, 1986;Lima and Coimbra, 1988;Burstein et al., 1990;Marshall et al., 1996;Li et al., 1996;Yu et al., 2005) and anterograde (Peschanski et al., 1983;LeDoux et al., 1987;Cliffer et al., 1991) tracing studies, together with early degeneration studies (Lund and Webster, 1967;Zemlan et al., 1978), have indicated that the rat spinothalamic tract originates from several parts of the spinal gray matter and terminates widely in the thalamus in regions that include VPL, Po, mediodorsal, centrolateral, parafascicular, and ventromedial nuclei. In some reports, specific populations of spinothalamic neurons were identified (Giesler et al., 1979;Dado and Giesler, 1990;Kobayashi, 1998), and these demonstrated that the lamina I component projects mainly to lateral thalamus. A recent anterograde study confirmed that neu-   rons in cervical superficial dorsal horn project to Po and VPL and also demonstrated a substantial projection to the caudal end of the thalamus (Gauriau and Bernard, 2004a). This latter projection extended from interaural ϳ3.2-4.4 mm and was centred on PoT.
Because PoT may not have been included in injection sites in previous studies, our first aim was to determine whether injections that included this region would label a larger number of lamina I spinothalamic neurons than previously reported. In the Thal series, injections included all thalamic regions known to receive significant input from superficial dorsal horn, including PoT (Gauriau and Bernard, 2004a). Although there was some variation between injections, numbers of retrogradely labeled lamina I neurons in C7 were very consistent, averaging 23.2 cells per 600 m. The mean number in L4 was considerably lower: 3.5 cells per 600 m. Because the length of C7 was 2.3 mm, whereas that of L4 is ϳ2.5 mm (Polgá r et al. 2004), we estimate that there are ϳ90 lamina I spinothalamic neurons on the contralateral side in C7 and 15 in L4. It is unlikely that this difference results from the longer transport distance from lumbar segments, because numerous lamina I neurons in the lumbar enlargement were labeled within a 3-day survival period following injections of CTb or Fluoro-Gold into CVLM, LPb, or PAG (e.g., Spike et al., 2003).
Two previous studies have quantified spinothalamic neurons in the rat: Lima and Coimbra (1988) estimated that there were 24 lamina I spinothalamic cells per side in C7 and 38 in L3-4, whereas Burstein et al. (1990) reported that there were 110 such cells in C7-8 and 19 in L4 -5. Our estimates are considerably higher than those in either of these studies for the C7 segment, but not for L4. The injections in these two previous studies did not apparently fill PoT consistently, which suggests that injecting tracer into PoT increases the number of labeled lamina I spinothalamic cells in the cervical, but not lumbar enlargement.
Surprisingly, the mean numbers of retrogradely labeled neurons in the PoT experiments were very similar to those for the Thal series. Although there was some spread of tracer into surrounding structures in these experiments, this did not involve areas known to receive input from the superficial dorsal horn, except as an extension of the terminal field centered on PoT (Gauriau and Bernard, 2004a). This suggests that injections targeted on PoT can label most (if not all) spinothalamic neurons in lamina I.
If the difference between numbers of labeled lamina I neurons in C7 in the present study and those reported previously is due to inclusion of PoT in our injections, this suggests that some lamina I cells in this segment project to PoT but no further rostrally. This was explored in the DI experiments (CTb injection into PoT, Fluoro-Gold injection into more rostral thalamic nuclei). If this interpretation was correct, we would expect to find that most lamina I cells in C7 were double-labeled or only contained CTb. The total numbers of spinothalamic lamina I cells in the DI experiments were similar to those in Thal and PoT experiments, whereas the mean number labeled with Fluoro-Gold in C7 was lower (Table 4). However, there was considerable variation in the numbers of Fluoro-Goldand CTb-labeled cells in this series, and cells labeled only with Fluoro-Gold were seen in significant numbers in some experiments. This may be explained partly by incomplete filling of PoT with CTb in some cases and may also result from the closeness of the Fluoro-Gold injection site to PoT resulting in suppression of CTb uptake, as it has been reported that injecting Fluoro-Gold proximal to other tracers in double-labeling experiments interferes with uptake of the second tracer (Akintunde and Buxton, 1992;Bice and Beal, 1997).
Although our results indicate that most lamina I spinothalamic neurons can be labeled by tracer injections into PoT, this might have resulted from uptake by fibers of passage, because the main bundle of spinothalamic axons passes through this region (LeDoux et al., 1987;Gauriau and Bernard, 2004a). Fluorescent latex microspheres are apparently not taken up by intact axons near the injection site (Pu and Amthor, 1990;Krug et al., 1998), and all three FLM experiments resulted in labeling of lamina I neurons in C6. In one experiment (FLM3) 46 of these cells were labeled and if the number of lamina I spinothalamic neurons in C6 is similar to that in C7, this would represent half of the spinothalamic population. Because the injection site in this animal occupied much less than half of the entire PoT, it is likely that this is a considerable underestimate. This suggests that most (if not all) lamina I spinothalamic neurons in the cervical enlargement have axons that arborize in or near PoT. Retrogradely labeled lamina I neurons were also seen in L5 in two of these experiments, and although the numbers were small, they represent approximately one-third of the expected population at this level. This indicates that some lamina I spinothalamic cells from the lumbar enlargement also project to PoT.

The proportion of lamina I neurons that project to the thalamus and their NK1r expression
The two methods that we used to estimate the proportion of lamina I neurons in C7 that belong to the spinothalamic tract gave slightly different values (2.4% and 1.9%). However, these are both much lower than the estimate by Yu et al. (2005), who concluded that 9% of lamina I neurons in the cervical enlargement were spinothalamic. It is difficult to explain the discrepancy between these results, although one significant methodological difference is the definition of the lamina I/II border in the two studies. We estimated the position of this border from the NK1r plexus whereas Yu et al. assumed a uniform width of 20 m for lamina I. We therefore recalculated the percentages based on the assumption that lamina I had a width of 20 m. Although this gave slightly higher values (3.2% and 2.1%), these are still far below that of Yu et al. (2005), and the method of placing the lamina I/II border therefore cannot account for these differences.
We did not directly determine the proportion of lamina I neurons in L4 that project to the thalamus, but this can be estimated from previously published data. Spike et al. (2003) used a similar disector method and concluded that there were 8,318 lamina I neurons per side in L4. However, in that study correction for tissue shrinkage was not performed, and this will therefore be an overestimate. In  Fig. 10. Injection sites for experiments FLM1-FLM3. Drawings show the spread of fluorescent latex microspheres (red) in these experiments. Each column represents a single experiment, and the experiment number (corresponding to those in Tables 1 and 8) is shown at the bottom of the column. Numbers at the top left of each drawing give the approximate position of the section anterior to the interaural plane. Drawings are based on those in Paxinos and Watson (2005). Eth, ethmoid thalamic nucleus; MGD, medial geniculate nucleus, dorsal part; MGV, medial geniculate nucleus, ventral part; other abbreviations as in Figure 1. The image below shows a darkfield photomicrograph (interaural ϳ3.9 mm) from experiment FLM3. Note the limited spread of the red fluorescent latex microspheres. Scale bar ϭ 1 mm in top and bottom panels. the present study the mean thickness of sections when scanned with the confocal microscope was found to be 45 m, corresponding to a shrinkage of 25%. The tissue used by Spike et al. was prepared in the same way and is therefore likely to have undergone similar shrinkage. If this correction is applied retrospectively to the data from Spike et al., the number of lamina I neurons per side in L4 would be 6,238. Because we found 15 spinothalamic lamina I cells in L4, these would constitute 0.2% of the neuronal population in this lamina. Marshall et al. (1996) reported that 77% of lamina I spinothalamic neurons in lumbar cord were NK1rimmunoreactive, and ϳ80% of lumbar lamina I neurons labeled from CVLM, LPb, or PAG express the receptor (Todd et al., 2000). Here, we obtained a similar result for spinothalamic lamina I neurons in L4 (84%) and C7 (87%). NK1r-immunoreactive lamina I projection neurons in lumbar cord receive strong monosynaptic input from substance P-containing (nociceptive) primary afferents (Todd et al., 2002), and the NK1r-expressing spinothalamic lamina I neurons presumably provide a powerful input from these afferents to the thalamus.

NK1r-expressing spinothalamic neurons in laminae III-IV
Lamina III/IV NK1r-immunoreactive neurons with long dorsal dendrites in lumbar cord also receive many synapses from substance P-containing afferents (Naim et al., 1997) and respond to various types of noxious stimulus (Polgá r et al., 2007). Most of these cells can be labeled from CVLM and around two-thirds project to LPb (Todd et al., 2000). The present results show that ϳ17% of those in L5 project to the thalamus, and this proportion is higher for cells located medially. Because the medial dorsal horn receives inputs from distal regions of the hindlimb (Swett and Woolf, 1985), distal limb inputs presumably have preferential access to the thalamus through spinotha-lamic cells of this type. Similar NK1r-immunoreactive cells have been identified in cervical cord (Brown et al., 1995), and we have found that these also receive numerous contacts from substance P-containing afferents (Al-Khater and Todd, unpublished observations). Here we show that the great majority of cells of this type in C6 belong to the spinothalamic tract.
In the DI experiments, very few of these cells in either C6 or L5 were labeled with Fluoro-Gold except in two cases (DI1 and 4) in which the Fluoro-Gold spread into rostral PoT. The other experiments in this series had extensive thalamic filling extending rostrally from interaural 4.6 mm, and including the VB complex, Po, and medial thalamus. The lack of Fluoro-Gold labeling in these experiments suggests that whereas these cells project to PoT, their axons do not arborize elsewhere in the thalamus. Zhang and Giesler (2005) identified spinothalamic neurons in laminae III-IV of rat cervical enlargement that projected only to caudal thalamus (mainly PoT and PIL), and their sample may have included some NK1r-expressing cells with long dorsal dendrites.
PoT projects to several areas, including S2 and insular cortices (Shi and Cassell, 1998;Linke and Schwegler, 2000), regions activated by painful stimuli in human imaging studies (Coghill et al., 1994;Casey et al., 1996). Gauriau and Bernard (2004b) demonstrated that rat PoT contains nociceptive-specific neurons that project to S2, as well as cells activated by noxious and tactile stimuli that project to insular cortex and amygdala. Most lamina I neurons are activated by noxious stimuli (Christensen and Perl, 1970;Bester et al., 2000), and these are likely to provide inputs to nociceptive-specific cells in PoT. Lamina III/IV NK1rimmunoreactive neurons receive monosynaptic inputs from myelinated (presumed low-threshold mechanoreceptive) primary afferents in lamina III, although these are much less numerous than those from substance Fig. 11. Retrograde labeling of lamina I neurons with fluorescent latex microspheres in parasagittal sections. The top row of images shows an example of a labeled lamina I neuron from C6 in experiment FLM3, and the bottom row shows a labeled neuron in L5 from experiment FLM2. In each case, separate images show the fluorescent latex microspheres (beads, red), NK1r (green) and NeuN (blue), with a merged image on the right. Note the presence of numerous beads in each neuron and that both neurons are NK1r-immunoreactive (arrow). Images are projections of seven (top row) or five (bottom row) confocal optical sections at 1-m z-spacing. Scale bar ϭ 20 m in bottom right panel (applies to all).